CN103145803A - Polypeptide in specific binding with breast cancer brain metastases cells - Google Patents

Abstract

The invention discloses a BrBP1 polypeptide that specifically binds to breast cancer brain metastases cells. The invention also relates to a method for preparing the polypeptide, which includes using phage display technology to perform multiple rounds of whole-cell subtractive screening to screen out the BrBP1 polypeptide that is associated with breast cancer brain metastases. Phage clones combined with cell lines; several phage clones were randomly selected, and strong positive phage clones were selected by cell ELISA method; strong positive phage clones were amplified and sequenced, and the measured BrBP1 polypeptide was synthesized artificially. The invention also relates to the application of the polypeptide in the preparation of medicines for treating breast cancer brain metastases and kits for early diagnosis of breast cancer brain metastases. The polypeptide can be synthesized artificially, and has small molecular weight, high activity, strong penetrating power, high affinity, high specificity, and low toxicity. It has good tumor targeting in vivo and in vitro, and can also be internalized into cells. It is suitable as a Vectors for targeted therapy.

Description

A polypeptide specifically binding to breast cancer brain metastasis cells

technical field

The present invention relates to a polypeptide for treating tumors, especially a polypeptide that specifically binds to breast cancer brain metastases cells. The present invention also relates to a method for preparing the polypeptide, and the polypeptide is used in the preparation of drugs for treating breast cancer brain metastases, The preparation is used in the early diagnosis kit of breast cancer brain metastases.

Background technique

Breast cancer is a malignant tumor that seriously endangers women's health. The morbidity and mortality of breast cancer worldwide rank first among female malignancies, accounting for 23% of all female cancer cases and 14% of cancer deaths. The typical manifestation of advanced breast cancer is distant spread and metastasis, mainly to bone and brain. Among them, the incidence of brain metastasis of breast cancer accounts for 10%-16% of metastatic cases. In recent years, with the prolongation of patient survival and the development of imaging diagnostic techniques, the detection rate of breast cancer brain metastases has increased year by year. However, existing drugs cannot effectively control and treat breast cancer brain metastases.

In recent years, the treatment of tumors with antibody drugs has received widespread attention. In the treatment of breast cancer, the monoclonal antibody drug Herceptin has a good effect on Her-2 positive metastatic breast cancer. For Her-2 positive breast cancer patients, radiotherapy combined with Herceptin is the standard treatment. However, the use of Herceptin before the diagnosis of distant metastases not only cannot effectively inhibit the occurrence of brain metastases, but also increases the relative risk of brain metastases by 1.57 times. According to reports, after the diagnosis of breast cancer with brain metastases, 50% of the patients were sensitive to chemotherapy and had stable metastatic symptoms, but half of the patients died of progressive brain metastases. The poor efficacy of Herceptin in the brain may be due to the low permeability of Herceptin to the blood-brain barrier. The level of Herceptin in the cerebrospinal fluid is only 1/420 of the injected dose, and the concentration increases to 1/49-1/76 after radiotherapy, but still cannot reach the effective therapeutic level.

Peptide drugs have small molecular weight, high activity, strong penetrating power, high affinity, high specificity, and low toxicity, which can overcome the above-mentioned problems in antibody therapy. At present, there is no peptide drug for the targeted treatment of breast cancer brain metastases. Therefore, the discovery of a polypeptide that can specifically bind to breast cancer brain metastases has extremely important application value for early diagnosis and effective treatment of breast cancer brain metastases. Patent CN101068935A discloses new human genes B1194, A2282V1, A2282V2 and A2282V3, whose expression is significantly increased in breast cancer. These genes and their encoded polypeptides can be used, for example, to diagnose breast cancer and as target molecules for the development of anti-breast cancer drugs . Patent CN101334411A discloses the screening, modification method, labeling and application of polypeptide drugs for the treatment of CXCR4 receptor-mediated breast cancer. Although both patent documents are polypeptides related to the treatment of breast cancer, neither of them mentioned that they can specifically bind to breast cancer brain metastases.

Contents of the invention

Purpose of the invention: Although it is mentioned in the background technology that there are patent documents related to polypeptides related to the treatment of breast cancer, there is no mention of polypeptides that can specifically bind to breast cancer brain metastases. The purpose of the present invention is to provide a It specifically binds to brain metastases cells, and also has good tumor targeting and can internalize polypeptides into cells.

Another object of the present invention is to provide a method for preparing a polypeptide specifically binding to breast cancer brain metastasis cells.

Another object of the present invention is to provide the application of the above polypeptide in the preparation of drugs for treating breast cancer.

Another object of the present invention is to provide the application of the above polypeptide in the preparation of a kit for early diagnosis of breast cancer brain metastases.

Technical solution: the purpose of the present invention is achieved through the following technical solutions.

The present invention prepares a polypeptide that specifically binds to breast cancer brain metastases cells, uses phage display technology, uses the breast cancer brain metastases cell line MDA-231BR as a positive screening cell, and uses the breast cancer parental cell line MDA-231P as a negative screening cell. Multiple rounds of whole-cell subtractive screening were performed to screen out phage clones that specifically bind to breast cancer brain metastases.

After phage monoclonalization, 120 phage clones were preliminarily identified by cell ELISA for their binding characteristics to cells, and strong positive clones with a P/N value greater than 3 were selected for sequencing. A total of 3 sequences were found in 22 phage clones that were successfully sequenced. The amino acid sequence of the dominant phage clone was named BrBP1. After sequencing, its gene sequence was shown as SEQ ID NO: 1. The measured BrBP1 sequence was artificially synthesized to obtain a BrBP1 polypeptide that specifically binds to breast cancer brain metastasis cells.

The present invention identifies the binding of phage clones to various cell lines by ELISA, confirms the specificity of binding of phage clones to breast cancer brain metastasis cells, and further identifies the binding specificity of phage clones to breast cancer brain metastasis cells through competition inhibition experiments.

The present invention also identifies the specificity of combining the phage clone with the target cell from the cell level in vitro and the level in vivo.

The present invention further confirms that the polypeptide can be internalized into cells, and the incubation time is dependent.

The polypeptide of the invention can be internalized into cells, can enter into tumor cells to kill target cells after being coupled with antitumor drugs, greatly enhances the antitumor effect, and can be used for preparing drugs for treating breast cancer.

The polypeptide described in the present invention can be used for the early diagnosis of breast cancer brain metastasis by preparing a kit through specific combination with breast cancer brain metastasis cells.

Beneficial effect: compared with prior art, the beneficial effect of the present invention has,

1. The present invention obtains a polypeptide that specifically binds to breast cancer brain metastases cells through screening, which fills the current gap of lack of polypeptides that bind to breast cancer brain metastases cells;

2. The polypeptide screened by the present invention is a small molecule polypeptide that can be synthesized artificially, with small molecular weight, high activity, strong penetrating power, high affinity, high specificity, and low toxicity, and is suitable as a carrier for targeted therapy;

3. The polypeptide screened by the present invention has good tumor targeting both in vitro and in vitro, laying a solid foundation for in-depth preclinical research on the polypeptide;

4. The polypeptide screened by the present invention can be internalized into cells, and after being coupled with anti-tumor drugs, it can enter into tumor cells to kill target cells, greatly enhancing the anti-tumor effect.

Description of drawings

Fig. 1 is the P/N ratio figure of 120 phage monoclonals detected by cell ELISA;

Figure 2 is a histogram comparing the affinity of Phage89 and VCSM13 phage clones with different tumor and normal cell lines detected by ELISA;

Figure 3 is a diagram of the experimental results of BrBP1-specific peptides and Control peptides competitively inhibiting the binding of Phage89 to MDA-231BR;

Figure 4 is the cellular immunofluorescence images of the binding of fluorescently labeled peptides BrBP1-K (5-TAMRA) and Control-K (5-TAMRA) to cell lines MDA-231BR and HL-7702 (magnification 400×);

Figure 5A is the flow cytometry results of different concentrations of BrBP1-K (5-TAMRA) combined with MDA-231BR;

Figure 5B is the flow cytometry results of different concentrations of Control-K (5-TAMRA) combined with MDA-231BR;

Figure 5C is a quantitative analysis of the percentage of positive cells combined with different concentrations of BrBP1-K (5-TAMRA), Control-K (5-TAMRA) and MDA-231BR in Figures 5A and 5B;

Figure 6 is a confocal microscope result of BrBP1 competitively inhibiting the binding of BrBP1-K (5-TAMRA) to MDA-231BR (magnification 400×);

Figure 7 is a graph showing the titer ratio of phage clones Phage89 and VCSM13 recovered from tumors and normal tissues and organs in the in vivo reinfusion experiment;

Figure 8 is near-infrared in vivo imaging images of near-infrared fluorescently labeled peptides BrBP1-K (Cy5.5) and Control-K (Cy5.5) at different time points;

Figure 9 is a diagram of the internalization of fluorescently labeled peptides BrBP1-K (5-TAMRA), Control-K (5-TAMRA) incubated with MDA-231BR for different times (magnification 400×).

Detailed ways

Used experimental material, main reagent and formula in the embodiment of the present invention are as follows:

Main experimental materials:

1. Cell lines: breast cancer brain metastases cell line MDA-231BR and breast cancer parental cell line MDA-231P are donated by the National Cancer Institute of the United States. Breast cancer cell line MCF-7, ovarian cancer cell line SK-OV-3, pancreatic cancer cell line PANC-1, gastric cancer cell line BGC-823, colon cancer cell line HT-29, prostate cancer cell line PC-3, liver cancer Cell line SMMC7721, human embryonic liver cell line HL-7702, human gastric mucosal immortalized cell line GES-1, and human embryonic kidney cell line 293T were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences;

2. Phage display 12 peptide library Ph.D.-12 and host strain ER2738: purchased from New England Biolabs;

3. Nude mice: purchased from the Academy of Military Medical Sciences.

Main reagents and formulas:

Main reagents:

1. Cell culture reagents: fetal bovine serum (GIBCO), DMEM medium (GIBCO), DMS (Sigma);

2. Main reagents for phage display peptide library screening: X-gal, DMF, NaN ₃ , Tween-20, BSA, glycine, and tetracycline were all purchased from Amresco; peptone and yeast extract were purchased from OXOID;

3. Main reagents for specific identification: HRP-labeled mouse anti-M13 antibody (GE Healthcare), Alexa Fluor 555-labeled goat anti-mouse antibody (Invitrogen).

Main reagent formula:

1. LB medium: Each liter contains 10g Bacto-Tryptone, 5g yeast extract, 5g NaCl. Autoclave, store at room temperature;

2. LB/IPTG/Xgal plate: LB medium + 15g/L agar powder. Autoclave, cool to below 70°C, add 1ml IPTG/Xgal, mix well and pour plate. Store the plate in the dark at 4°C;

3. Top agar: Each liter contains 10g Bacto-Tryptone, 5g yeast extract, 5g NaCl, 1g MgCl ₂ ·6H ₂ O, 7g agar powder. Autoclave and divide into 50ml aliquots. The solid medium is stored at room temperature and melted in a microwave oven when used;

4. Tetracycline stock solution: dissolve in 50% ethanol at a concentration of 20mg/ml, store at -20°C in the dark, and shake well before use;

5. LB-Tet plate: LB medium + 15g/L agar powder. Autoclave, when cooled to below 70°C, add 1ml of tetracycline stock solution, mix well and pour plate, and store the plate in the dark at 4°C;

6. Blocking buffer: 0.1M NaHCO ₃ (pH 8.6), 5mg/ml BSA, 0.02% NaN ₃ , sterilized by filtration, stored at 4°C;

7. TBS: 50mM Tris-HCl (pH 7.5), 150mM NaCl, autoclaved, stored at room temperature;

8. PBS: Contain 8.00g of NaCl, 0.20g of KCl, 3.58g of Na ₂ HPO ₄ 12H ₂ O, 0.24g of KH ₂ PO ₄ , PH=7.2, autoclave, store at room temperature;

9. PEG/NaCl: 20% (w/v) PEG-8000, 2.5M NaCl, autoclaved, stored at room temperature;

10. Iodide buffer: 10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI. Store at room temperature away from light;

11. IPTG/X-gal formula: Dissolve 1.25g of IPTG (isopropyl β-D-thiogalactoside) and 1g of X-gal in 25ml of dimethylformamide. Store the solution at -20°C away from light;

12. TMB chromogenic solution:

Liquid A: Contains 18.41g Na ₂ HPO ₄ 12H ₂ O, 5.10g citric acid, 0.60g urea hydrogen peroxide per liter, pH=5.0;

Solution B: EDTA·2Na 0.146g, citric acid 0.15g dissolved in 800ml distilled water, add TMBC (0.1gTMB+10ml absolute ethanol) drop by drop, dilute to 1L;

Working solution: Mix liquid A and liquid B at a ratio of 1:1, keep away from light, and prepare immediately after use.

Screening and preparation of embodiment 1 BrBP1 polypeptide

The present invention screens and prepares BrBP1 polypeptide through the following methods and steps.

1. Preliminary screening of phage clones that bind to breast cancer brain metastases cell lines

Phage display technology was used to conduct multiple rounds of whole-cell subtractive screening, and the phage clones that combined with the breast cancer brain metastases cell line MDA-231BR were preliminarily screened out. The specific steps are as follows:

In the first round of screening, only the breast cancer brain metastases cell line MDA-231BR was used for positive screening. Breast cancer brain metastases cell line MDA-231BR cells were digested with trypsin, adjusted the cell density to 2.5×10 ⁵ /ml, seeded in cell culture dishes (60×15mm ³ ), and waited for the cells to grow to 80%-90% confluence , filter.

The specific steps of screening are as follows:

(1) Sealing: Use a pipette to suck out the medium in the above-mentioned MDA-231BR cell culture dish, wash the cells with 1×TBS for 3 times, and then add serum-free DMEM to continue culturing for 2 hours. Then remove serum-free DMEM, add 0.5% [m/v]BSA/TBS blocking solution, and block at 37°C for 1h;

(2) Incubation: remove the blocking solution, add 1×10 ¹¹ pfu phage 12 peptide library, shake gently at 37°C for 1 hour;

(3) Washing: Remove the supernatant and wash 6 times with TBST (TBS+0.1%[v/v]Tween-20) buffer;

(4) Elution: use non-specific elution buffer 0.2M Glycine-HCl (PH 2.2), 1mg/ml BSA at room temperature for 10min, gently shake at room temperature, add 150ul1M Tris-HCl (PH 9.1) to neutralize the eluate after collection;

(5) Amplification: After the eluate was measured, the remaining eluate was added to the 20ml ER2738 culture in the early logarithmic growth stage for amplification, which was used for the next round of screening.

In the second round of screening, the parental breast cancer cell line MDA-231P was used for negative screening, and then the breast cancer brain metastases cell line MDA-231BR was used for positive screening. In the incubation phase, the secondary library of the first round of screening was incubated with MDA-231P for 1 hour, washed twice, and the supernatant and washing liquid were added to MDA-231BR for incubation for 1 hour. At the same time, in this round of screening, the concentration of Tween-20 in the negative sieve washing solution TBST was 0.5%, the Tween-20 concentration in the positive sieve washing solution TBST was 0.3%, and the positive sieve was washed 8 times.

In the third round of screening, the parental breast cancer cell line MDA-231P was used for negative screening, and then the breast cancer brain metastases cell line MDA-231BR was used for positive screening. In the incubation phase, the secondary library of the second round of screening was first incubated with MDA-231P for 1 hour, and after washing once, the supernatant and washing liquid were added to MDA-231BR for incubation for 1 hour. At the same time, in this round of screening, the concentration of Tween-20 in the negative sieve washing solution TBST was 0.3%, the Tween-20 concentration in the positive sieve washing solution TBST was 0.5%, and the positive sieve was washed 10 times.

In the fourth round of screening, the parental breast cancer cell line MDA-231P was used for negative screening, and then the breast cancer brain metastases cell line MDA-231BR was used for positive screening. In the incubation phase, the secondary library of the third round of screening was first incubated with MDA-231P for 1 hour, and then the supernatant was added to MDA-231BR for incubation for 1 hour. At the same time, in this round of screening, the concentration of Tween-20 in the negative sieve washing liquid TBST was 0.1%, the Tween-20 concentration in the positive sieve washing liquid TBST was 0.5%, and the positive sieve was washed 10 times.

The titer of the eluate from the fourth round of panning was determined on LB/IPTG/Xgal plate. Pick 120 blue phage plaques from the eluate titer plate obtained from the fourth round of screening with a sterilized toothpick or tip, and transfer them to a 1 ml ER2738 culture tube in the pre-logarithmic growth stage to amplify the monoclonal phage.

According to the titer of the phage eluate in each round of screening, the yield and enrichment were calculated. Yield = the number of recovered phages/the number of input phages. Enrichment rate = nth round of screening yield/n-1th round of screening yield (n≥2).

The selection pressure and enrichment of the whole screening process are shown in Table 1. In the four rounds of screening, the yield of each round was improved to varying degrees compared with the previous round. The total enrichment of phage clones was 43.9 times.

Table 1 Selection pressure control and enrichment of phage clones for peptide screening combined with breast cancer brain metastases cell lines

2. Preliminary identification of phage-positive clones by cell ELISA

In the above experiments, 120 phage clones were randomly selected, and the binding level of 120 phage clones to the breast cancer brain metastases cell line MDA-231BR was preliminarily identified by cell ELISA method.

MDA-231BR was seeded in a 96-well plate at a density of 1×10 ⁴ /well, cultured for 24 hours, treated without serum for 1 hour, washed with PBS three times, and then fixed with 4% paraformaldehyde/PBS at room temperature for 20 minutes. Add 2% skimmed milk powder/PBS to block for 1 h, then add 1×10 ¹⁰ pfu phage monoclonal and incubate at 37°C for 1 h. After washing 6 times, add HRP-labeled mouse anti-M13 antibody and incubate at 37°C for 1h. After washing 6 times, add 100ul/well TMB to develop color for 10min, add 2M H ₂ SO ₄ to terminate the reaction, and detect the OD450nm absorbance with a microplate reader. In this experiment, empty phage VCSM13 without any foreign insert was used as a negative control. The experiment was set up with 3 replicate wells and repeated 3 times.

The experimental results are shown in Figure 1. The number of phage single clones in the figure is the number of 120 randomly selected phage single clones, P/N ratio=OD450nm value of phage single clone combined with MDA-231BR/VCSM13 combined with MDA-231BR For the OD450nm value, Figure 1 visually expresses the P/N ratio in shades of gray, and the darker the gray, the larger the P/N. It can be seen from Figure 1 that different phage clones have different degrees of affinity to MDA-231BR. Among these 120 clones, strong positive clones with P/N≥3 were selected for the next experiment.

3. DNA extraction and sequencing

After phage clone amplification, centrifuge to remove bacteria, take 500ul phage supernatant and add 200ul PEG/NaCl, mix well and let stand at room temperature for 10min, centrifuge at 14000rpm/min at 4°C for 10min, resuspend the precipitate in 100ul iodide buffer thoroughly, add 250ul After incubating with ethanol for 10 minutes at room temperature, centrifuge for 10 minutes, discard the supernatant, wash the pellet with 70% ethanol, dry it briefly in vacuum, and resuspend the pellet in 30ul TE for sequencing.

The sequencing of this experiment was completed by Huada Genomics. The sequencing primer was 5'- ^HO GTA TGG GAT TTT GCT AAA CAA C-3'.

The results of phage clone sequencing were analyzed by SeqMan software, and the base sequence was translated into amino acid sequence according to the genetic code table. According to statistics, the 22 phage clones successfully sequenced have 3 sequences, among which 16 phage clones carry foreign insert sequences as shown in SEQ ID NO: 1, accounting for 72.7%, named BrBP1.

4. Synthesis of BrBP1 polypeptide

The BrBP1 polypeptide was artificially synthesized by Gill Biochemical (Shanghai) Co., Ltd. according to the above amino acid sequence. The synthetic product was purified by high-performance liquid chromatography (HPLC) and identified by mass spectrometry (MS). The purity of the synthetic polypeptide was 98%. %above.

Bioinformatics analysis of embodiment 2 BrBP1 polypeptide

In this example, the ProtParam tool (http://web.expasy.org/protparam/) provided by the ExPASy website was used to analyze the physical and chemical properties of the above-mentioned BrBP1 polypeptide. As shown in Table 2, the molecular weight of the polypeptide is 1487.6, the theoretical isoelectric point is 4.00, it is composed of 199 atoms, the extinction coefficient is 5.700, and the half-life in mammalian reticulocytes is about 30h. is 32.50, and the overall average hydrophobicity is -0.858.

Table 2 Analysis of physical and chemical characteristics of BrBP1

Example 3 The specificity of BrBP1 polypeptide binding to breast cancer brain metastases cell lines

1. Identify the combination of phage clones and various cell lines by ELISA

In this experiment, the No. 89 phage clone (named Phage89) carrying the BrBP1 fragment was selected, and the binding ability of Phage89 to different cell lines was identified by ELISA. Breast cancer brain metastasis cell MDA-231BR, breast cancer parental cell line MDA-231P, breast cancer cell line MCF-7, ovarian cancer SK-OV-3, pancreatic cancer PANC-1, gastric cancer BGC-823, colon cancer HT- 29. Prostate cancer PC-3, liver cancer SMMC7721 cells, human embryonic liver cell line HL-7702, human gastric mucosal immortalized cell line GES-1, and human embryonic kidney cell line 293T were digested with trypsin at a density of 1×10 ⁴ /well Inoculated in 96-well plates, cultured for 24 hours, treated without serum for 1 hour, washed with PBS three times, and then fixed with 4% paraformaldehyde/PBS at room temperature for 20 minutes. Add 2% skimmed milk powder/PBS to block for 1 h, then add 1×10 ¹⁰ pfu phage monoclonal and incubate at 37°C for 1 h. After washing 6 times, add HRP-labeled mouse anti-M13 antibody and incubate at 37°C for 1h. After washing 6 times, add 100ul/well TMB to develop color for 10min, add 2M H ₂ SO ₄ to terminate the reaction, and detect the OD450nm absorbance with a microplate reader. In this experiment, empty phage VCSM13 was used as a negative control. The experiment was set up with 3 replicate wells and repeated 3 times.

The experimental results are shown in Figure 2. Phage89 has a high binding level to the breast cancer brain metastasis cell line MDA-231BR, but a low binding level to the parental breast cancer cell line MDA-231P, and a low spontaneous metastasis cell line MCF-7 Does not bind to ovarian cancer cell line SK-OV-3, pancreatic cancer cell line PANC-1, gastric cancer cell line BGC-823, colon cancer cell line HT-29, prostate cancer cell line PC-3, liver cancer cell line SMMC7721 No binding, no binding to human embryonic liver cell line HL-7702, human gastric mucosa immortalized cell line GES-1, human embryonic kidney cell line 293T. This result indicated that the screened phage clones targeted breast cancer cell lines, especially with a higher level of binding to brain metastasis cell lines.

2. Competitive inhibition experiments further identified the specificity of phage clones binding to breast cancer brain metastasis cells

The BrBP1 polypeptide and its control peptide Control (GGGAGGGAGGGA) were chemically synthesized for the competition inhibition experiment of polypeptide-phage clones.

Breast cancer brain metastasis cells MDA-231BR were trypsinized and inoculated in a 96-well plate at a density of 1×10 ⁴ /well, cultured for 24 hours, treated for 1 hour without serum, washed with PBS for 3 times, and then washed with 4% paraformaldehyde/ Cells were fixed with PBS at room temperature for 20 min. Add 2% skimmed milk powder/PBS to block for 1h. Dilute BrBP1 and Control peptide 2 times from 1×10 ^-4 mol/L to 14 gradients, add to the sealed 96-well plate and incubate with MDA-231BR cells at 37°C for 1 hour, then add 1×10 ¹⁰ pfu phage monoclonal Phage89 and empty phage VCSM13 were incubated at 37°C for 1h. After washing 6 times, add HRP-labeled mouse anti-M13 antibody and incubate at 37°C for 1h. After washing 6 times, add 100ul/well TMB to develop color for 10min, add 2M H ₂ SO4 to terminate the reaction, and detect the absorbance at OD450nm with a microplate reader. The experiment was set up with 3 replicate wells and repeated 3 times.

The results of polypeptide-phage clone competition inhibition experiment are shown in Figure 3. BrBP1 and Control peptides were diluted 14 gradients by 2 times from 1×10 ^-4 mol/L. Phage clones gradually increased, and the OD450nm value gradually increased. The IC50 of BrBP1 half inhibitory rate was calculated to be 6.83×10 ^-6 mol/L. When the peptide concentration drops below 1.95×10 ^-7 mol/L (2 ^-9 dilution level), the inhibitory effect on the combination of Phage89 and MDA-231BR is already very weak, almost zero. However, with the change of Control peptide concentration, the binding level of Phage89 and MDA-231BR remained unchanged, indicating that Control peptide had no effect on the binding of Phage89 and MDA-231BR.

Example 4 The specificity of BrBP1 polypeptide binding to breast cancer brain metastases cell lines in vitro

In this example, after the BrBP1 polypeptide was chemically synthesized and labeled with 5-TAMRA fluorescence, the binding affinity and specificity of the polypeptide to MDA-231BR were identified in vitro.

1. Polypeptide fluorescent labeling

The BrBP1 polypeptide and its control peptide Control (GGGAGGGAGGGA) were chemically synthesized. A lysine K was added to the C-terminus of the BrBP1 and Control peptides, which was connected to the 5-TAMRA fluorescent dye through its side chain hydroxyl. Labeled fluorescent BrBP1 and Control peptides are denoted as BrBP1-K (5-TAMRA) and Control-K (5-TAMRA), respectively. The chemical synthesis of the above peptides was completed by Jill Biochemical (Shanghai) Co., Ltd., purified by high-performance liquid chromatography (HPLC), and identified by mass spectrometry (MS). The purity of all 5-TAMRA fluorescently labeled peptides is above 98%.

2. BrBP1 polypeptide binding ability to different cell lines

Breast cancer brain metastasis cells MDA-231BR and human embryonic liver cell line HL-7702 were trypsinized and inoculated in 24-well plates at a density of 5×10 ⁴ /well. After culturing for 24 hours, they were washed 3 times with PBS, and then added 1 ×10 ^-6 mol/L BrBP1-K (5-TAMRA) and Control-K (5-TAMRA) were incubated at 37°C for 20min. After washing three times, the cells were fixed with 4% paraformaldehyde/PBS at room temperature for 20 min. Wash 3 times, add DAPI to stain cell nuclei at 37°C for 15 min, wash 3 times and observe under an inverted fluorescence microscope. The experiment was set up with 3 replicate wells and repeated 3 times. Among them, the Abs/Em=555/565nm of the fluorescent dye 5-TAMRA, and the Abs/Em=350/457nm of DAPI.

Figure 4 is the fluorescence diagram of the binding of BrBP1-K (5-TAMRA), Control-K (5-TAMRA) and MDA-231BR, HL-7702. A is the fluorescent image of the binding of fluorescently labeled peptide BrBP1-K (5-TAMRA) to the cell line MDA-231BR, B is the fluorescent image of the binding of fluorescently labeled peptide Control-K (5-TAMRA) to the cell line MDA-231BR, and C is the fluorescent image The fluorescence image of the binding of the labeled peptide BrBP1-K (5-TAMRA) to the cell line HL-7702, D is the fluorescence image of the binding of the fluorescently labeled peptide Control-K (5-TAMRA) to the cell line HL-7702, showing that BrBP1-K (5- TAMRA) can bind to MDA-231BR but not to HL-7702, indicating that its binding is specific.

3. Flow cytometry proves the specificity of BrBP1 polypeptide binding to breast cancer brain metastases cell lines in vitro

Breast cancer brain metastasis cells MDA-231BR were digested with trypsin, adjusted to a cell concentration of 1×10 ⁵ /ml, washed with PBS for 3 times, and then added with 0.25×10 ^-6 mol/L, 1×10 ^-6 mol/L, 4×10 ^-6 mol/L, 16×10 ^-6 mol/L BrBP1-K (5-TAMRA) and Control-K (5-TAMRA) were incubated at 37°C for 1 hour. After washing 3 times, check on the machine. The experiment was repeated three times.

The results of this experiment are shown in Figures 5A, 5B, and 5C, in which Figures 5A and 5B are the flow cytometry results of different concentrations of BrBP1-K (5-TAMRA) and Control-K (5-TAMRA) combined with MDA-231BR . It can be seen from Figure 5A that as the concentration of BrBP1-K (5-TAMRA) increases, the percentage of positive cells increases, and Figure 5B shows that the concentration of Control-K (5-TAMRA) increases, but there are still no positive binding cells. Figure 5C is a graph showing the percentage of positive cells changing with the concentration of fluorescent peptides. It can be seen that as the concentration of BrBP1-K (5-TAMRA) increases, the percentage of positive MDA-231BR cells bound to BrBP1-K (5-TAMRA) increases. When it was 16×10 ^-6 mol/L, the percentage of positive cells was 64.21%. The calculated dissociation constant of BrBP1-K (5-TAMRA) binding to MDA-231BR cells was Kd=0.9344×10 ^{- 6} mol/L. And Control-K (5-TAMRA) does not bind to MDA-231BR.

4. Competitive inhibition experiments further proved the specificity of BrBP1 polypeptide binding to breast cancer brain metastases cell lines in vitro

In this experiment, confocal microscopy was used for imaging, and it was divided into three groups: the experimental group (1×10 ^-6 mol/L BrBP1-K (5-TAMRA)), the control group (1×10 ^-6 mol/L Control-K (5-TAMRA)), competition group (1×10 ^-6 mol/L BrBP1-K (5-TAMRA)+1×10 ^-5 mol/LBrBP1). The main steps are as follows: breast cancer brain metastases MDA-231BR cells were trypsinized and inoculated on laser confocal culture dishes at a density of 5×10 ⁴ /well, cultured for 24 hours, treated without serum for 1 hour, washed with PBS for 3 times, and then respectively Add the experimental group, control group and BrBP1 competition group and incubate at 37°C for 20min. After washing three times, the cells were fixed with 4% paraformaldehyde/PBS at room temperature for 20 min. Wash 3 times, add DAPI to stain cell nuclei at 37°C for 15 min, wash 3 times and observe under confocal laser microscope. The experiment was repeated 3 times. Among them, the Abs/Em=555/565nm of the fluorescent dye 5-TAMRA, and the Abs/Em=350/457nm of DAPI.

The results of this experiment are shown in Figure 6, where A is the binding of the experimental group polypeptide (1×10 ^-6 mol/L BrBP1-K (5-TAMRA)) to MDA-231BR, B is the competition group polypeptide (1× 10 ^-6 mol/L BrBP1-K (5-TAMRA) + 1×10 ^-5 mol/L BrBP1) combined with MDA-231BR. When the concentration of BrBP1 was 10 times (1×10 ^-5 mol/L) that of BrBP1-K (5-TAMRA), BrBP1 could completely competitively inhibit the binding of BrBP1-K (5-TAMRA) to MDA-231BR. The above results fully illustrate the specificity of BrBP1-K (5-TAMRA) binding to MDA-231BR.

Example 5 Specificity of BrBP1 Polypeptide Binding to Breast Cancer Brain Metastasis Cell Lines in Vivo

1. In vivo reinfusion experiment to study the biological distribution of phage clones carrying BrBP1 polypeptide fragments in tumor-bearing mice

Construction of tumor-bearing mice: the nude mice in this patent were purchased from the Academy of Military Medical Sciences, and they were all 4-5 week old female mice. Nude mice were kept in the SPF grade animal room of Southeast University. After the MDA-231BR cells were digested with trypsin, the cell concentration was adjusted to 5×10 ⁵ /200ul/mouse, and inoculated subcutaneously on the right side of the back of the nude mice. The tumor formation in nude mice was observed every two days and the tumor volume was measured. After 2-3 weeks, the tumor volume reached 300-500 mm ³ and was used for the experiment. Tumor volume = 1/2 length × width ² .

In vivo reinfusion experiment: inject 1×10 ¹¹ pfu bacteriophage into the tail vein, anesthetize with ether after 15 minutes of circulation, perfuse more than 200ml of PBS in the right ventricle, dissect, separate nude mouse tumors, heart, liver, lung, kidney, brain, and weigh the tissue homogeneously slurry, releasing the phage. Determination of phage titers. Calculate and compare the titer values of phage per unit mass of tissue. In this experiment, the experimental group was injected with Phage89 (No. 89 phage clone carrying the BrBP1 fragment), and the control group was injected with empty phage VCSM13. Both the experimental group and the control group had 4 nude mice.

Calculate the ratio of phage titers per unit mass of tumor tissue to unit mass of other tissues, and draw a graph. As shown in Figure 7, the ratio of the titer value of Phage89 per unit mass (g) of tumor tissue to the titer value per unit mass (g) of heart, liver, lung, kidney, and brain was 12.21, 3.48, 4.85, 3.30, 31.43, that is, the titer of tumor tissue is 2.30-30.43 times higher than that of other tissues and organs. The ratios of the titer values of empty phage VCSM13 in unit mass (g) of tumor tissue to unit mass (g) of heart, liver, lung, kidney, and brain were 0.85, 0.87, 0.82, 0.66, and 18.46, respectively. From the above results, it can be seen that no matter Phage89 or VCSM13, the number of phages entering the brain tissue is much lower than that of tumors and other tissues, which may be related to the blood-brain barrier restricting the entry of phages. The titer of Phage89 in tumor tissue is higher than that in other normal tissues, but not in VCSM13, which fully demonstrates that Phage89 carrying the BrBP1 insertion fragment has tumor targeting, while VCSM13 does not.

2. Identification of BrBP1 polypeptide specificity in vivo by near-infrared in vivo imaging

In this experiment, BrBP1 and Control peptides were labeled with fluorescein Cy5.5, which were denoted as BrBP1-K (Cy5.5) and Control-K (Cy5.5). The above synthetic peptides were completed by Sangon Bioengineering (Shanghai) Co., Ltd. The synthetic products were purified by high performance liquid chromatography (HPLC), and identified by mass spectrometry (MS). The purity of all Cy5.5 fluorescently labeled peptides was above 95%.

A nude mouse model bearing breast cancer brain metastases was constructed and used for experiments when the tumor volume grew to 300-500 mm ³ . This experiment was divided into two groups: the experimental group (injection of 1nmol of BrBP1-K (Cy5.5) into the tail vein) and the control group (injection of 1nmol of Control-K (Cy5.5) in the tail vein). The experimental steps are as follows: the experimental group and the control group were injected with fluorescently labeled polypeptides through the tail vein, and after isoflurane gas anesthesia, the mice were fixed on the CRi Maestro multispectral in vivo imaging system (CRi, Woburn, MA, USA), and detected for 30 min and 60 min respectively. imaging. Among them, Abs/Em=673/707nm of fluorescent dye Cy5.5.

The results of this experiment are shown in Figure 8. At 30 min and 60 min, the fluorescence signal of BrBP1-K (Cy5.5) at the tumor site was significantly higher than that of the control group Control-K (Cy5.5) at the tumor site. In the experimental group, the liver also had a strong fluorescent signal at 30 minutes, but as time went on, the fluorescent signal at the tumor site at 60 minutes was much higher than that of other tissues and organs. This shows that BrBP1-K (Cy5.5) has good tumor targeting in vivo and in vivo.

Example 6 BrBP1 polypeptide incubation time dependence and cell internalization research

In this example, the incubation time of the polypeptide and MDA-231BR was further extended to 1 h, and the level of internalization of the polypeptide into the cells at different incubation times was analyzed. The specific implementation steps are as follows:

Breast cancer brain metastasis cells MDA-231BR were trypsinized and seeded in 24-well plates at a density of 5×10 ⁴ /well. After culturing for 24 hours, they were washed with PBS three times, and then 1×10 ^-6 mol/L BrBP1 was added respectively. -K (5-TAMRA) and Control-K (5-TAMRA) were incubated at 37°C for 30min, 45min, and 60min. After washing three times, the cells were fixed with 4% paraformaldehyde/PBS at room temperature for 20min. Wash 3 times, add DAPI to stain cell nuclei at 37°C for 15 min, wash 3 times and observe under an inverted fluorescence microscope. The experiment was set up with 3 replicate wells and repeated 3 times. Among them, the Abs/Em=555/565nm of the fluorescent dye 5-TAMRA, and the Abs/Em=350/457nm of DAPI.

The results of this experiment are shown in Figure 9, where A-C are the fluorescence images of the internalization of BrBP1-K (5-TAMRA) and MDA-231BR incubated for 30 min, 45 min, and 60 min, respectively. As the incubation time prolongs, BrBP1-K (5-TAMRA) TAMRA) is gradually internalized into cells. After incubation for 30 minutes, the staining of the cell membrane was mainly observed, while at 45 minutes, the cytoplasm had already appeared obvious staining, and at 60 minutes, the cell nucleus had also appeared obvious staining. D-F are the staining results of Control-K (5-TAMRA) incubated with MDA-231BR for 30min, 45min, and 60min. It can be seen that Control-K (5-TAMRA) does not bind to MDA-231BR. Therefore, BrBP1-K (5-TAMRA) polypeptide can be internalized into cells in a time-dependent manner.

sequence listing

<110> Southeast University

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<120> A polypeptide specifically binding to breast cancer brain metastasis cells

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<170>PatentIn version 3.5

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Met Tyr Pro Trp Thr Glu Pro Ser Tyr Leu Ser Asn

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Claims (4)

1. polypeptide with breast cancer patients with brain transitional cell specific binding, called after BrBP1, its aminoacid sequence is as shown in SEQ ID NO:1.

2. method for preparing the polypeptide of claimed in claim 1 and breast cancer patients with brain transitional cell specific binding is characterized in that carrying out according to the following steps:

(1) adopt display technique of bacteriophage, carry out the full cell of many wheels and subdue screening, preliminary screening goes out the phage clone of being combined with the breast cancer patients with brain transfer cell line;

(2) choose at random several phage clones, the phage clone of picking out by cell ELISA method preliminary evaluation and breast cancer patients with brain transfer cell line in conjunction with level, and select the strong positive phage clone to carry out next step experiment;

(3) will check order after above-mentioned strong positive phage clone amplification processing, wherein a kind of aminoacid sequence of phage clone is BrBP1 polypeptide claimed in claim 1;

(4) above-mentioned BrBP1 polypeptide is synthesized by manual type.

3. the polypeptide of claimed in claim 1 and breast cancer patients with brain transitional cell specific binding is in the application for the preparation for the treatment of breast cancer patients with brain diversion medicaments.

4. the polypeptide with breast cancer patients with brain transitional cell specific binding claimed in claim 1 is shifting the application of the test kit of early diagnosis for the preparation of breast cancer patients with brain.

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